ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 14, NUMBER 9, 2005

Cell Transplantation, Vol. 14, pp. 609-620, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation

Takeshi Aoki,1 Tomotake Koizumi,1 Yasuna Kobayashi,2 Daisuke Yasuda,1 Yoshihiko Izumida,1 Zhenghao Jin,1 Nobukazu Nishino,1 Yoshinori Shimizu,1 Hirohisa Kato,1 Noriyuki Murai,1 Takashi Niiya,1 Yuta Enami,1 Keitaro Mitamura,1 Toshihiro Yamamoto,1 and Mitsuo Kusano,1

1Second Department of Surgery, School of Medicine, Showa University, Tokyo, Japan
2Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37°C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Key words: Hepatocyte transplantation; Microencaspsulation; Cryopreservation; Alginate/poly-L-lysine

Address correspondence to Takeshi Aoki, M.D., Ph.D., Second Department of Surgery, School of Medicine, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, Japan. Tel: 81-3-3784-8541; Fax: 81-3-3784-5835; E-mail: takejp@wb4.so-net.ne.jp




Cell Transplantation, Vol. 14, pp. 621-627, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Stability and Repeat Regeneration Potential of the Engineered Liver Tissues Under the Kidney Capsule in Mice

Kazuo Ohashi,1,2 Mark A. Kay,2 Takashi Yokoyama,1 Hiroyuki Kuge,1 Hiromichi Kanehiro,1 Michiyoshi Hisanaga,1 Saiho Ko,1 and Yoshiyuki Nakajima1

1Department of Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan
2Departments of Pediatrics and Genetics, Stanford University Medical Center, 300 Pasteur Dr., Stanford, CA 94305, USA

Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 ± 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.

Key words: Tissue engineering; Hepatocyte transplantation; Liver regeneration; Liver failure; Regenerative medicine

Address correspondence to Kazuo Ohashi, M.D., Ph.D., First Department of Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Tel: 81-744-22-3051, ext. 3419; Fax: 81-744-24-6866; E-mail: kohashi@naramed-u.ac.jp or Mark A. Kay, M.D., Ph.D., Department of Pediatrics, Stanford University Medical Center, Rm G-305, 300 Pasteur Dr., Stanford, CA 94305, USA. Tel: 650-498-6531; Fax: 650-498-6540; E-mail: markay@stanford.edu




Cell Transplantation, Vol. 14, pp. 629-635, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Long-Term Maintenance of Liver-Specific Functions in Cultured ES Cell-Derived Hepatocytes With Hyaluronan Sponge

Takumi Teratani,1 Gary Quinn,1 Yusuke Yamamoto,1,2 Tomoya Sato,1 Hiroko Yamanokuchi,3 Akira Asari,3 and Takahiro Ochiya1

1National Cancer Center Research Institute, 1-1, Tsukiji, 5-chome, Chuo-ku, Tokyo 104-0045, Japan
2Department of Biology, School of Education, Waseda University, Tokyo 169-0051, Japan
3Central Research Laboratories, Seikagaku Corporation, 1253 Tateno 3-chome, Higashiyamato-shi, Tokyo 207-0021, Japan

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.

Key words: Embryonic stem cells; Hepatocytes; Hyaluronan (HA) sponge; Three-dimensional; Spheroid

Address correspondence to Takahiro Ochiya, Ph.D., Head of Section for Studies on Metastasis, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Tel: 81-3-3542-2511; Fax: 81-3-3541-2685; E-mail: tochiya@ncc.go.jp




Cell Transplantation, Vol. 14, pp. 637-645, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

PDX-1 Protein Is Internalized by Lipid Raft-Dependent Macropinocytosis

Hirofumi Noguchi,1 Shinichi Matsumoto,1 Teru Okitsu,1 Yasuhiro Iwanaga,1 Yukihide Yonekawa,1 Hideo Nagata,1 Masayuki Matsushita,2 Fan-Yan Wei,2 Hideki Matsui,2 Kohtaro Minami,3 Susumu Seino,3 Yumi Masui,4 Shiroh Futaki,4,5 and Koichi Tanaka1

1Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
2Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
3Department of Experimental Therapeutics, Translational Research Center, Kyoto University Graduate School of Medicine, 54 kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
4Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
5PRESTO, JST, Kawaguchi, Saitama, Japan

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell-surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4-64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4-64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Key words: PDX-1; Protein transduction system; Protein transduction domain; Lipid raft macropinocytosis; Endocytosis

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Transplantation and Immunology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, Japan 606-8507. Tel: 81-75-751-4699; Fax: 81-75-751-3896; E-mail: noguchih@kuhp.kyoto-u.ac.jp




Cell Transplantation, Vol. 14, pp. 647-653, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Differentiation of Adult Hepatic Stem-Like Cells Into Pancreatic Endocrine Cells

Satoko Yamada,1 Kunihiko Terada,2 Yasuharu Ueno,2 Toshihiro Sugiyama,2 Masaharu Seno,3 and Itaru Kojima1

1Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
2Department of Biochemistry, Akita University School of Medicine, Akita 010-8543, Japan
3Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan

To apply cell transplantation for treatment of diabetes mellitus, a sufficient number of β-cell sources are required. In the present study, we examined whether an epithelial cell line obtained from normal adult rat liver, namely hepatic stem-like (HSL) cells, which can be converted to both hepatocytes and billiary epithelial cells, could be a potential b-cell source. The growth speed of HSL cells was rapid and these cells were easily expanded in vitro. Bipotential hepatic stem cells, HSL cells, also expressed PGP9.5, which is expressed in neurons, b-cells, and progenitor cells of the pancreatic endocrine cells as well. Sodium butyrate induced morphological changes in HSL cells and converted them into flattened cells with large cytoplasm. When HSL cells were incubated with a combination of 5 mM sodium butyrate and 1 nM betacellulin, most of the cells were converted into morphologically neuron-like cells. RT-PCR analysis revealed that a series of transcriptional factors involved in differentiation of pancreatic endocrine cells was induced by the treatment with sodium butyrate and betacellulin. mRNAs for insulin, pancreatic polypeptide, and somatostatin were also observed. Immunoreactive pancreatic polypeptide, somatostatin, and insulin were detected in sodium butyrate and betacellulin-treated HSL cells. In conclusion, HSL cells obtained from adult normal liver also have the potential to differentiate into pancreatic endocrine cells in vitro. HSL cells may be one of the potential b-cell sources for cell transplant therapy for insulin-dependent diabetes.

Key words: Hepatic stem-like cell; Liver epithelial cell; Endocrine cell differentiation; Insulin

Address correspondence to Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. Tel: 81-27-220-8835; Fax: 81-27-220-8893; E-mail: ikojima@showa.gunma-u.ac.jp




Cell Transplantation, Vol. 14, pp. 655-663, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vivo PET Measurements With [11C]PE2I to Evaluate Fetal Mesencephalic Transplantations to Unilateral 6-OHDA-Lesioned Rats

Motoki Inaji,1,2 Takahito Yoshizaki,3,5 Takashi Okauchi,1 Jun Maeda,1 Yuji Nagai,1 Tadashi Nariai,2 Kikuo Ohno,2 Kiyoshi Ando,4 Hideyuki Okano,3,5 Shigeru Obayashi,1 and Tetsuya Suhara1

1Brain Imaging Project, National Institute of Radiological Science, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
2Department of Neurosurgery, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
3Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
4Neurobehavioral Unit, Central Institute for Experimental Animal, Kawasaki 216-0001, Japan
5Japanese Scientific and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo, Japan

Positron emission tomography (PET) is a useful tool to assess and visualize neurotransmissions in vivo. In this study, we performed repeated PET scans with [11C]PE2I, a tracer of the dopamine transporter, to evaluate the alteration of the expression of dopamine (DA) transmission component after a fetal mesencephalic transplantation. The fetal mesencephalic cells were transplanted into the striatum of unilateral 6-OHDA-lesioned rats. PET scans with [11C]PE2I were performed to evaluate the DA transporter before and 2 and 4 weeks after the transplantation. Rotation behavior tests, in vitro autoradiography, measurements of DA contents in the striatum by high-performance liquid chromatography (HPLC), and tyrosine hydroxylase (TH) immunohistological examinations were performed at the same time points and examined for their relationship to changes in the dopamine transporter. The number of ipsilateral rotations induced by methamphetamine injections decreased. DA contents in the striatum measured with HPLC significantly increased. In the PET study, the binding potential of [11C]PE2I increased at 4 weeks. The results of the in vitro autoradiography study corresponded with those of the PET study. The degrees of the change in the binding potentials correlated with those of the numbers of rotations in the behavioral study and the DA contents in the striatum. In the histological examination, TH-positive cells with axons were observed at 2 and 4 weeks after the transplantation. As the dopamine transporter exists only in the axon terminal of DA neurons, these results suggested that PET measurements of [11C]PE2I binding indicated not only survival, but maturity and functioning of the transplanted cells. Repeated PET measurements of DA transporters are a useful tool in assessing the effectiveness of neural transplantations.

Key words: PET; PE2I; Transplantation; Dopamine transporter; 6-Hydroxydopamine (6-OHDA)

Address correspondence to Dr. Tetsuya Suhara, Brain Imaging Project, National Institute of Radiological Science, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. Tel: 81-43-206-3194; Fax: 81-43-253-0396; E-mail: suhara@nirs.go.jp




Cell Transplantation, Vol. 14, pp. 665-672, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Promotion of Neurite Outgrowth From Fetal Hippocampal Cells by TNF-a Receptor 1-Derived Peptide

Kazumi Kajiwara, Shin-ichi Ogata, and Masao Tanihara

Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

Cytokines such as tumor necrosis factor-a (TNF-a), FasL, and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis or inflammation through binding to their specific receptors, TNFR1, Fas, and DR5, respectively. We have previously reported ligand-binding and cell death-inhibiting synthetic peptides, which were designed based on the crystal structure of a ligand-receptor complex and the homology of the amino acid sequence among the death receptor family members. Here we show that, among these death receptor-derived peptides, the TNFR1-derived peptide specifically arrested cell proliferation and promoted cell adhesion of fetal rat (E16) hippocampal cells, and promoted neurite outgrowth from hippocampus-derived neurospheres cultured with the addition of the peptide or cultured on a peptide-coated surface. Furthermore, among these death receptor-derived peptides, marked neurite outgrowth was observed only when the neurospheres were cultured on a TNFR1-derived peptide-conjugated covalently cross-linked alginate gel. The neurites from the neurospheres positively immunostained with an antibody against neurofilaments. These results suggest that the TNFR1-derived peptide promotes neuronal differentiation of the hippocampal neural stem cells and the TNFR1-derived peptide-conjugated covalently cross-linked alginate gel may be a useful material for assisting neural stem cell transplantation.

Key words: Tumor necrosis factor receptor 1 (TNFR1); Neural stem cells; Synthetic peptide; Differentiation; Alginate gel

Address correspondence to Prof. Masao Tanihara, Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Tel: 81-743-72-6120; Fax: 81-743-72-6129; E-mail: mtanihar@ms.naist.jp




Cell Transplantation, Vol. 14, pp. 673-682, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vitro Screening of Exogenous Factors for Human Neural Stem/Progenitor Cell Proliferation Using Measurement of Total ATP Content in Viable Cells

Yonehiro Kanemura,1,4* Hideki Mori,1* Atsuyo Nakagawa,1* Mohammed Omedul Islam,1,2 Eri Kodama,1 Atsuyo Yamamoto,1  Tomoko Shofuda,1 Satoshi Kobayashi,1 Jun Miyake,1 Tomohiko Yamazaki,1,3 Shun-ichiro Hirano,1,2 Mami Yamasaki,4,5 and Hideyuki Okano6

1Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan
2Institute of Biomedical Research and Innovation, Kobe, Hyogo 650-0047, Japan
3New Energy and Industrial Technology Development Organization, Tokyo 170-6028, Japan
4Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan
5Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan
6Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

Key words: Human neural stem/progenitor cell; ATP assay; Neuropshere; Culture condition

Address correspondence to Yonehiro Kanemura M.D., Ph.D., Institute for Clinical Research, Osaka National Hospital, Hational Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka city, Osaka 540-0006, Japan. Tel: +81-6-6942-1331; Fax: +81-6-6496-3530; E-mail: kanemura@onh.go.jp

*These three authors contributed equally to this work.




Cell Transplantation, Vol. 14, pp. 683-693, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Synergistic Effects of FGF-2 With Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes

Tsuguharu Takahashi,1,2 Toru Ogasawara,1,3 Junji Jusguniti,4 Guangyao Liu,1,5 Hirotaka Asato,6 Takashi Nakatsuka,7 Eijyu Uchinuma,8 Kozo Nakamura,4 Hiroshi Kawaguchi,4 Ung-il Chung,2 Tsuyoshi Takato,2,3 and Kazuto Hoshi1,2

1Department of Fujisoft ABC Cartilage & Bone Regeneration, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
2Division of Tissue Engineering, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
3Department of Oral & Maxillofacial Surgery, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
4Department of Clinical Bioinformatics, Graduate School of Medicine and Faculty of Medicine,The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
5Department of Orthopaedics Surgery, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
6Department of Plastic & Reconstructive Surgery, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
7Department of Plastic & Reconstructive Surgery, Saitama Medical School, Kerohongo 38, keroyama-cho, Iruma, Saitama 350-0495, Japan
8Department of Plastic & Reconstruction Surgery, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor(FGF)-2, insulin-like growth factor(IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1a25-dihydroxy vitamin D3, L-3,3´,5´-triodothyronine, interleukine-1 receptor antagonist, 17b-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2 , while the cell number synergistically gained a 10-12-fold increase with 5 mg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 mg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

Key words: Chondrocyte; Proliferation; Regenerative medicine; Medium; Soluble factor; Fractional factorial design

Address correspondence to Kazuto Hoshi, M.D., Ph.D., Department of Fujisoft ABC Cartilage & Bone Regeneration, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-8655, Japan. Tel: 81-3-3815-5411, ext. 37386; Fax: 81-3-5800-9891; E-mail: pochi-tky@umin.ac.jp




Cell Transplantation, Vol. 14, pp. 695-700, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Time Course Evaluation of Reparative Cartilage With MR Imaging After Autologous Chondrocyte Implantation

Atsuya Watanabe,1,2 Yuichi Wada,1 Takayuki Obata,2 Takahisa Sasho,1 Takuya Ueda,3 Mitsuru Tamura,2 Hiroo Ikehira,2 and Hideshige Moriya1

1Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, Chiba, 260-8677 Japan
2National Institute of Radiological Sciences, Chiba, 263-8555 Japan
3Department of Radiology, Graduate School of Medicine, Chiba University, Chiba, 260-8677 Japan

The aim of this study was to evaluate the qualitative change in reparative cartilage after autologous chondrocyte implantation (ACI). Ten knees of 10 patients were studied. The signal intensities of reparative and normal cartilage were evaluated by fat-suppressed three-dimensional spoiled-gradient recalled (FS 3D-SPGR) MR imaging. The signal intensity (SI) index (signal intensity of reparative cartilage divided by that of normal cartilage) was defined and the change in SI index was investigated. Histological and biochemical evaluation was done at the second look arthroscopy. The SI index was at its lowest level immediately after ACI and increased with time to 9 months thereafter. After 9-12 months, the SI index settled to almost level and was maintained at that value for at least 2-3 years postoperatively. The average of the SI indexes after 12 months to the last examination was 74.2 ± 4.6 (range 64.2-82.8), which means signal intensity of reparative cartilage was maintained at a value lower than that of normal cartilage. The total ICRS score was 11.6 ± 2.3 points (mean ± SD). The GAG concentration was 107.9 ± 17.0 mg/mg (mean ± SD) in normal cartilage and 65.9 ± 9.4 mg/mg in reparative cartilage. The quality of reparative cartilage as hyaline cartilage was inferior to that of normal cartilage. In the present study, the time course change in the SI index indicates that the major maturation process of implanted chondrocytes neared completion in 9-12 months. Minor changes, such as matrix remodeling with reorganization of the collagen fibers in reparative cartilage, may continue, but an almost identical condition seemed to be maintained during the first 2-3 years of follow-up. SI index does not always reflect all properties of reparative cartilage but may be a useful parameter for noninvasive evaluation.

Key words: Autologous chondrocyte implantation; Magnetic resonance imaging; Time course evaluation; Signal intensity; Reparative cartilage

Address correspondence to Atsuya Watanabe, Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-Ku, Chiba-Shi, 260-8677 Japan. Tel: 81-43-226-2117; Fax: 81-43-226-2116; E-mail: atsuyan1@pa2.so-net.ne.jp




Cell Transplantation, Vol. 14, pp. 701-708, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Phenotype-Specific Cells With Proliferative Potential Are Produced by Polyethylene Glycol-Induced Fusion of Mouse Embryonic Stem Cells With Fetal Cardiomyocytes

Shunsuke Takei,1 Makoto Yamamoto,1 Li Cui,1 Fengming Yue,1 Kohei Johkura,1 Naoko Ogiwara,1 Hisae Iinuma,2 Kota Okinaga,2 and Katsunori Sasaki1

1Department of Anatomy and Organ Technology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
2Department of Surgery, Teikyo University School of Medicine, Tokyo, Japan

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.

Key words: Mouse embryonic stem cells; Cardiomyocytes; Cell fusion; Polyethylene glycol; Proliferation

Address correspondence to Shunsuke Takei, Department of Anatomy and Organ Technology, Shinshu University School of Medicine, 3-3-3 Asahi, Matsumoto 390-8621, Japan. Tel: 81-263-37-2590; Fax: 81-263-37-3093; E-mail: takei@sch.md.shinshu-u.ac.jp




Cell Transplantation, Vol. 14, pp. 709-714, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Fetal Dermal But Not Loose Fascial Mesenchymal Cells Possess Regenerative Activity of Dermal Structure

Kazuo Kishi,1 Kenichi Katsube,2 Hiroko Satoh,3 Nobuaki Imanishi,4 Hideo Nakajima,1 and Tatsuo Nakajima1

1Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjukuku, Tokyo, 160-8582 Japan
2Molecular Pathology, Graduate School of Tokyo Medical and Dental University, Tokyo, 113-8549 Japan
3Department of Plastic and Reconstructive Surgery, National Tokyo Medical Center, Tokyo, 152-8902 Japan
4Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi Shinjukuku, Tokyo, 160-8582 Japan

Fetal skin possesses a regenerative activity until certain developmental stages. However, the origin of cells that regenerate dermis after wounding has not been clarified yet. In the present study we located the origin of cells that reconstruct fetal dermal structure by histological examination and by marking cells in the loose fascia. Next we evaluated the regenerative activity of fetal dermal mesenchymal cells by cotransplanting with fetal epidermal cells onto the skin defect of scid mice. We conclude that fetal dermal mesenchymal cells but not loose fascial cells possess regenerative activity even on the environment in scid mice.

Key words: Fetal wound healing; Regeneration; scid mouse; Dermal

Address correspondence to Dr. Kazuo Kishi, Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjukuku Tokyo, 160-8582 Japan. Tel: 81-(0) 3-3352-1524; Fax: 81-(0) 3-3352-1054; E-mail: kkishi@sc.itc.keio.ac.jp